Structure And Function Analysis Of Antibody By Random Mutation Library Of CDR3

Monoclonal antibodies are the largest and fastest growing group of therapeutic proteins.At present,more than 70 kinds of antibody drugs have been approved by clinical medicine.In 2017,the market size of antibody drugs exceeded 100 billion US dollars.At present,incomplete statistical results show that 500 therapeutic antibodies and their derivatives are in clinical trials,mainly in cancer treatment and autoimmune diseases.The advent of antibody drugs has brought hope for some major diseases,and with the development of the application,the monoclonal antibodies of large molecules to some extent have limited their application because of their own structural problems.This problem has greatly stimulated scientists’enthusiasm for antibody modification,especially the miniaturization of antibodies.NBL42 is a nanobody which only has CDR3 and two sides frame area.The antibody has high specific binding activity and significant enzyme inhibitory activity to lysozyme.Analysis of the antibody sequence revealed that the CDR3 length was 17 amino acids,and some amino acid residues in the frame region were different from those of the currently known camel source VHH.Therefore,it is envisaged that nanobody NBL42 can be used as a model to study the relationship between antibody structure and function.The main purpose of this study is two aspects:first,using the NBL42-ΔCDR3random mutation library to analyze the distribution of amino acid in CDR3 area of nanobody.Secondly,the method of site directed mutation and transplantation is used to verify whether the NBL42 frame area can be used as the general skeleton of the mutant library or the framework of CDR transplantation;for the further design of the library,To provide reference for the miniaturization of antibody,it will provide some experimental basis for the design of new antibody in the future.The first part is to analyze the distribution of amino acids in the area of nanobody CDR3 by using NBL42-CDR3 random mutation library.First,the overlapping PCR technique was used to preserve the frame region(FR3-FR4)of the NBL42 nanobody,and the 17 amino acids in the CDR3 region were randomly mutated according to the mutation rule of NNY.The NBL42-Delta CDR3random mutation DNA fragment was obtained by gene synthesis.The NBL42-ΔCDR3 library was constructed with a storage capacity of 4.8*10~7.Then two antigens(lysozyme and CD47 recombinant protein)were used to screen the mutant library for multiple rounds.After the screening was completed,polyclonal Phage ELISA was used to verify the enrichment of the library.The results showed that the library was enriched by multiple rounds of screening,and 100 monoclonal antibodies were selected from the last two rounds of screening,and 3 nanobodies against lysozyme were obtained.The antibody sequence was subcloned to the expression vector pET22b(+)to induce expression and purification to obtain the antibody protein.The result of indirect ELISA showed that the affinity of the 3 nanobodies was low.In order to further understand the distribution of amino acid in the CDR3 region of the nanobody,we analyzed the high throughput sequencing data of the unimmunized camel VHH,including 1209159 sequences,and the length of the CDR3region was 6-33 amino acids.The frequency of the occurrence of amino acids is calculated,and the frequency of the statistics is analyzed with the chi square analysis of the frequency of the theory.The conservativeness of each site is confirmed,and some loci in the CDR3 region are found to be conservative.These loci exist at both ends of the CDR3 region,and the 105 and 106 bits are mostly alanine and 107 bits of ammonia.The basic acids are glycine,aspartic acid and lysine;the 108 amino acids are arginine,proline and alanine;tyrosine at 115 and 117.Compared with the NBL42-ΔCDR3 random mutation library CDR sequence,it was found that the amino acid distribution and conservatism of the mutant library were significantly different from those of the natural library,indicating that the mutant library had a lower functional VHH diversity.The second part analyzes the framework area of NBL42 by means of transplantation and site directed mutation.First,the FR3 and FR4 region of the nanobody NBL42,which is screened from the immune library,is used as a receptor,and the CDR3 region of VHH H5 of PD1’s VHH H5 is used as a donor to synthesize NBL42-H5 CDR3 recombinant nanobody DNA fragment and connect to the pET22b expression vector to express and purify the recombinant nanobody;the ELISA method is detected.Its combination with PD1.The results showed that NBL42-H5 CDR3 was successfully constructed to pET22b(+)and successfully expressed NBL42-H5 CDR3 recombinant nanobody.ELISA results showed that the antibody NBL42-H5 CDR3 after transplantation had VHH H5 partial antigen binding activity.The same method selected a short peptide CERX(a polypeptide of SIRP alpha specifically),and transplants its 12 amino acid into the framework of NBL42 to form a recombinant peptide antibody and construct the target gene on the pET22b(+)expression vector.ELISA results showed that the antibody NBL42-CERX after transplantation still had strong antigen binding activity.We used site directed mutagenesis to synthesize the amino acid mutation and gene synthesis of eighty-eighth NBL42 nanobodies.The gene was synthesized by the Kingsy biotechnology company in Nanjing.The target fragment was connected to the pET22b(+)expression vector to be expressed and purified,and the nanobody NBL42-C88Y was obtained.The binding activity of the nano antibody to lysozyme was verified by ELISA.The final ELISA results showed that the binding activity of nano antibody NBL42-C88Y and lysozyme showed that the binding ability of C88Y mutated nanoscale antibody to lysozyme was significantly lower than that of NBL42.